Coordinate induction of AMP deaminase in human atrium with mitochondrial DNA deletion

Despite the heteroplasmic lower population of mitochondrial (mt) DNA deletion, mtDNA deletion is significantly related to the loss of atrial adenine nucleotides. To elucidate its mechanism, we examined the frequency of a 7.4-kb mtDNA deletion, the concentration of adenine nucleotides, and the activity of AMP catabolic enzymes in 10 human right atria obtained from cardiac surgery, using quantitative PCR, HPLC, and immunoprecipitations. The atrial concentrations of ATP, ADP, AMP, and the total adenine nucleotides were significantly lower in patients with deletion than those in patients without deletion, despite the lower frequency of their deletion. The activities of total AMP deaminase (AMPD), liver-type (AMPD 2), and heart-type isoform (AMPD 3) were significantly higher in patients with deletion than in patients without deletion, although there was no significant difference in the cytosolic 5(')-nucleotidase among them. In conclusion, mtDNA deletion coordinately induces AMP deaminase to contribute to the loss of atrial adenine nucleotides through degrading AMP excessively.     

Phosphorylation of retinoblastoma-related protein by the cyclin D/cyclin-dependent kinase complex is activated at the G1/S-phase transition in tobacco

In mammals, D-type cyclin-associated kinases mainly regulate the G1/S transition by phosphorylating the retinoblastoma (Rb) protein. We previously demonstrated that in tobacco, cyclin D (Nicta; CycD3;3) is complexed with the PSTAIRE-containing cyclin-dependent kinase (CDKA) from tobacco. Here, we show that Nicta; CycD3;3-associated kinases phosphorylate both the tobacco Rb-related protein (NtRb1) and histone H1. Although NtRb1 kinase activity was detected only during the middle G1- to early S-phase, histone H1 kinase activity was observed as two peaks in G1- to S-phase and G2/M- to M-phase. Importantly, we show that the proportion of cells in the G1-phase was reduced in transgenic Bright Yellow-2 cells overexpressing Nicta; CycD3;3-GFP. Mutational analyses revealed that phosphorylation of Thr-191 in Nicta; CycD3;3 possibly is required for both full kinase activity and localization predominantly to the nucleus. These data suggest that Nicta; CycD3;3 acts as a rate-limiting regulator in the G1/S transition by forming active complexes with CDKA or its related kinases to phosphorylate Rb-related protein and potentially plays a novel role during G2/M and mitosis.     

Methamphetamine-induced hyperactivity and behavioral sensitization in PACAP deficient mice

Mice lacking the PACAP gene (PACAP(-/-)) display psychomotor abnormalities such as novelty-induced hyperactivity and jumping behavior, and they show different responses to amphetamine, a typical psychostimulant. The present study examined the possible role of endogenous PACAP in methamphetamine (METH)-induced hyperactivity and behavioral sensitization. The locomotor activity of hyperactive PACAP(-/-) mice was measured using the infrared photocell beam detection system, Acti-Track, after a habituation period. Single administration of METH (1 and 2mg/kg) caused a robust increase in locomotor activity of mice, but this effect did not differ between wild-type and PACAP(-/-) mice. Repeated administration of METH (1mg/kg) for 7 days enhanced METH-induced hyperactivity, and this sensitization was observed even when withdrawn for 7 days. There was no difference in the degree of development and expression of METH-induced behavioral sensitization between wild-type and PACAP(-/-) mice. In addition, there was no difference in METH-induced increases in extracellular serotonin and dopamine levels in the prefrontal cortex of the normal and sensitized mice between the two groups. These results suggest that endogenous PACAP is not involved in the locomotor stimulant activity of acute METH and repeated METH-induced behavioral and neurochemical sensitization.     

Unmasking of CD22 Co-receptor on Germinal Center B-cells Occurs by Alternative Mechanisms in Mouse and Man

CD22 is an inhibitory B-cell co-receptor whose function is modulated by sialic acid (Sia)-bearing glycan ligands. Glycan remodeling in the germinal center (GC) alters CD22 ligands, with as yet no ascribed biological consequence. Here, we show in both mice and humans that loss of high affinity ligands on GC B-cells unmasks the binding site of CD22 relative to naive and memory B-cells, promoting recognition of trans ligands. The conserved modulation of CD22 ligands on GC B-cells is striking because high affinity glycan ligands of CD22 are species-specific. In both species, the high affinity ligand is based on the sequence Siaα2–6Galβ1–4GlcNAc, which terminates N-glycans. The human ligand has N-acetylneuraminic acid (Neu5Ac) as the sialic acid, and the high affinity ligand on naive B-cells contains 6-O-sulfate on the GlcNAc. On human GC B-cells, this sulfate modification is lost, giving rise to lower affinity CD22 ligands. Ligands of CD22 on naive murine B-cells do not contain the 6-O-sulfate modification. Instead, the high affinity ligand for mouse CD22 has N-glycolylneuraminic acid (Neu5Gc) as the sialic acid, which is replaced on GC B-cells with Neu5Ac. Human naive and memory B-cells express sulfated glycans as high affinity CD22 ligands, which are lost on GC B-cells. In mice, Neu5Gc-containing glycans serve as high affinity CD22 ligands that are replaced by Neu5Ac-containing glycans on GC B-cells. Our results demonstrate that loss of high affinity CD22 ligands on GC B-cells occurs in both mice and humans through alternative mechanisms, unmasking CD22 relative to naive and memory B-cells.     

Melanin biosynthesis inhibitors from Tarragon Artemisia dracunculus

The EtOH extract of tarragon Artemisia dracunculus, a perennial herb in the family Asteraceae, was found to potently inhibit α-melanocyte-stimulating hormone (α-MSH) induced melanin production in B16 mouse melanoma cells. Bioassay-guided fractionation led to the isolation of two alkamide compounds, isobutyl (1) and piperidiyl (2) amides of undeca-2E,4E-dien-8,10-dynoic acid. The respective EC(50) values for melanin biosynthesis inhibition were 1.8 and 2.3 μg/mL for 1 and 2.     

Position-specific incorporation of dansylated non-natural amino acids into streptavidin by using a four-base codon

Novel non-natural amino acids carrying a dansyl fluorescent group were designed, synthesized, and incorporated into various positions of streptavidin by using a CGGG four-base codon in an Escherichia coli in vitro translation system. 2,6-Dansyl-aminophenylalanine (2,6-dnsAF) was found to be incorporated into the protein more efficiently than 1,5-dansyl-lysine, 2,6-dansyl-lysine, and 1,5-dansyl-aminophenylalanine. Fluorescence measurements indicate that the position-specific incorporation of the 2,6-dnsAF is a useful technique to probe protein structures. These results also indicate that well-designed non-natural amino acids carrying relatively large side chains can be accepted as substrates of the translation system.     

No influence of tumor growth by intramuscular injection of hepatocyte growth factor plasmid DNA: safety evaluation of therapeutic angiogenesis gene therapy in mice

Recently, a novel therapeutic treatment for ischemic diseases using angiogenic growth factors to augment collateral artery development has been proposed. As intramuscular injection of naked human hepatocyte growth factor (HGF) plasmid DNA induced therapeutic angiogenesis in several animal test subjects, we have started a clinical trial to treat peripheral arterial disease. However, one might assume that over-expression of angiogenic growth factors could enhance tumor growth. To resolve this issue, we examined the over-expression of HGF in tumor bearing mice. Tumors on their backs were prepared with an intradermal inoculation of A431, human epidermoid cancer cells expressing c-Met. These mice were intramuscularly injected with human HGF plasmid or control plasmid into the femoral muscle. Human HGF concentration was increased only in the femoral muscle, but not in blood. Although recombinant HGF stimulated the growth of A431 cells in vitro, temporally and locally HGF elevation in hindlimb had no effect on tumor growth in mice.